Role of (1,3)(1,4)-β-Glucan in Cell Walls: Interaction with Cellulose

Mar 28, 2014 - Oligosaccharide profiling using endo-(1,3)(1,4)-β-glucanase indicated that there was no difference in the frequency and distribution o...
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Role of (1,3)(1,4)-β-Glucan in Cell Walls: Interaction with Cellulose Sarah N. Kiemle,*,† Xiao Zhang,‡ Alan R. Esker,‡ Guillermo Toriz,§,⊥ Paul Gatenholm,§,∥ and Daniel J. Cosgrove† †

Department of Biology, Pennsylvania State University, University Park, Pennsylvania 16803, United States Department of Chemistry, Macromolecules and Interfaces Institute, Virginia Tech, Blacksburg, Virginia 24061, United States § Wallenberg Wood Science Center, Kemivägen 4, Gothenburg SE-412 96, Sweden ∥ Department of Chemical and Biological Engineering, Chalmers University of Technology, Kemivägen 4, Gothenburg SE-412 96, Sweden ⊥ Department of Wood, Cellulose and Paper Research, University of Guadalajara, Juárez 976, Guadalajara 44100, Mexico ‡

S Supporting Information *

ABSTRACT: (1,3)(1,4)-β-D-Glucan (mixed-linkage glucan or MLG), a characteristic hemicellulose in primary cell walls of grasses, was investigated to determine both its role in cell walls and its interaction with cellulose and other cell wall polysaccharides in vitro. Binding isotherms showed that MLG adsorption onto microcrystalline cellulose is slow, irreversible, and temperature-dependent. Measurements using quartz crystal microbalance with dissipation monitoring showed that MLG adsorbed irreversibly onto amorphous regenerated cellulose, forming a thick hydrogel. Oligosaccharide profiling using endo-(1,3)(1,4)-β-glucanase indicated that there was no difference in the frequency and distribution of (1,3) and (1,4) links in bound and unbound MLG. The binding of MLG to cellulose was reduced if the cellulose samples were first treated with certain cell wall polysaccharides, such as xyloglucan and glucuronoarabinoxylan. The tethering function of MLG in cell walls was tested by applying endo-(1,3)(1,4)-β-glucanase to wall samples in a constant force extensometer. Cell wall extension was not induced, which indicates that enzyme-accessible MLG does not tether cellulose fibrils into a load-bearing network.

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INTRODUCTION

linkages. Approximately 30% of the linkages are (1,3), with the remainder being (1,4). 4 Oligosacchraide profiling after digestion with endo-(1,3)(1,4)-β-glucanase shows MLGs of grasses to be composed of a mixture of trisaccharide and tetrasaccharide units, with each unit containing two or three contiguous (1,4)-β-glucosyl residues joined by a single (1,3) βlinkage.4,5 Longer blocks with more than three (1,4)-linked residues constitute up to 10% of the polymer.6 Contiguous (1,3)-linked glycosyl residue units have not been found.7 The (1,3)-β-glucosyl linkages create kinks within the linear polymer, thus creating an irregular shape2a that increases flexibility and solubility. In the aleurone and starchy endosperm of grains, including barley, wheat, and oat, MLG is found in high concentrations and is thought to act as a storage carbohydrate.2b,8 MLG has also been identified in the cell walls of coleoptiles9 and young leaves10 of grasses. Because its abundance (10−20% of the cell wall mass) in these tissues is highest prior to maximal growth rate and nearly disappears as cell expansion ends, MLG has been hypothesized to play a role in cell expansion.2b,11

Plant cell walls consist of cellulose microfibrils embedded in a matrix of polysaccharides and glycoproteins. The composition of the matrix and the connections between its various components are largely responsible for the differences in mechanical properties that occur in different types of plant cells. The composition of the cell wall matrix also influences the pulping properties of wood fibers, the strength and flexibility of lumber and textiles, the health effects of dietary fibers, and cell wall recalcitrance to deconstruction for biofuel production.1 Thus, there is much to gain from understanding the relationship between the mechanics and the organization of the cell wall. The composition of the cell wall varies between tissue types and phylogenetic groups. In the Poaceae (the grass family), pectin and xyloglucan (XyG) are present only in small amounts,2 whereas they are abundant in primary cell walls of most other land plants. The predominant noncellulosic matrix polysaccharides in primary cell walls of grasses are arabinoxylans (also called glucuronoarabinoxylan or GAX) and (1,3)(1,4)-β-D-glucan (mixed-linkage glucan or MLG). MLGs are considered to be a defining feature of the grass cell wall.2b,3 MLGs are unbranched and unsubstituted chains of βglucopyranosyl monomers linked through (1,3) and (1,4) © 2014 American Chemical Society

Received: January 24, 2014 Revised: March 25, 2014 Published: March 28, 2014 1727

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was quantified by phenol/sulfuric acid assay18 with glucose as the standard. The amount of bound sugar was calculated as the difference between the initial amount added and the amount remaining in the supernatant. To assess binding inhibition by other cell wall polysaccharides, 1 mg of Avicel was preincubated with 100 μg of MLG, XyG, AX, arabinan, or galactan, each diluted to a final volume of 1 mL with sodium acetate buffer (20 mM, pH 5.5). The samples were agitated on a thermoshaker at 1500 rpm at 60 °C for 4 h and then centrifuged for 10 min at 13 000g, and the supernatant was removed. Ten to two-hundred fifty migrograms of the second polysaccharide (MLG, XyG, AX, arabinan, or galactan) in 1 mL of sodium acetate buffer (20 mM, pH 5.5) was added to the pellet of Avicel and prebound polysaccharide. The samples were agitated again for 4 h at 60 °C and then centrifuged for 10 min at 13 000g, and the sugar content of the supernatant was assessed as described above. In addition to the phenol/sulfuric acid assay, monosaccharide analysis was performed on the supernatant of the binding-inhibition samples to determine displacement of the prebound polysaccharide or addition of the second polysaccharide to the prebound polysaccharide and Avicel. The monosaccharide analysis protocol was derived from Fry et al.19 and Hardy et al.20 In brief, 200 μL of sample supernatant, 140 μL of ddH2O, and 60 μL of concentrated trifluoroacetic acid were mixed, incubated at 121 °C for 2 h, cooled on ice, filtered with 0.2 μm Millex-LG syringe-driven filter units (cat. no. SLLGR04NL), and analyzed by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC−PAD) on a CarboPac PA20 column at a flow rate of 0.5 mL/min (Dionex ICS-5000 capillary reagent-free IC system). The column was first eluted with 10 mM sodium hydroxide for 5 min and then ramped to 100 mM sodium hydroxide over 35 min. Oligosaccharide Characterization. The oligosaccharide composition of the MLG bound to the Avicel substrate was compared with the unbound MLG in solution using the following experiments. Samples of MLG bound to Avicel (35 μg/1 mg) were washed extensively with 20 mM sodium phosphate (pH 6.6) to remove MLG in the supernatant; 1 mL of 20 mM sodium phosphate was added, vortexed, and centrifuged (13 000g for 5 min). This was repeated three times. The bound and unbound MLG in solution (35 μg MLG in 1 mL of 20 mM sodium phosphate, pH 6.6) were digested with 20 U (1 unit of activity is the amount of enzyme required to release 1 μmole of D-glucose-reducing-sugar equivalents per minute) of endo-(1,3)(1,4)β-glucanase for 4 h at 40 °C. Enzymatic digests were heated at 95 °C for 5 min, cooled on ice, and filtered with a 0.20 μm Millex-LG filter (cat. no. SLLGR04NL). The digests were analyzed by HPAEC−PAD on a CarboPac PA200 column at a flow rate of 0.5 mL/min. The HPAEC−PAD was stabilized with 100 mM sodium hydroxide buffer for 9 min followed by gradient of elution, ramping over 25 min to 100 mM sodium hydroxide with 100 mM sodium acetate buffer. Quartz Crystal Microbalance with Dissipation Monitoring (QCM-D). RC was deposited on QCM-D gold sensors (QSX-301; QSense). The sensors were cleaned with UV/ozone ProCleaner (Bioforce Nanoscience, Inc.) for 20 min and boiled in a mixture of ammonium hydroxide, hydrogen peroxide, and water (1:1:5 v/v) for 20 min. The sensors were then rinsed exhaustively with ultrapure water prior to spin-coating. TMSC solutions (10 g/L of TMSC in toluene) were spin-coated onto cleaned sensors at 2000 rpm for 1 min. The TMSC solutions were prepared by dissolution of 0.1 g of TMSC powder in 10 mL of toluene and filtered with a Teflon filter (pore size = 0.45 μm). Trimethylsilyl groups were removed by exposure of the coated surfaces to the vapor of a 10% (w/w) aqueous solution of hydrochloric acid for 5 min, yielding a smooth RC film with thickness of ∼10 nm and root-mean-square (rms) roughness of ∼1 nm.17,21 The sensor, coated with RC film, was placed into the QCM-D flow cell (E4 system; Q-Sense). Water was introduced into the flow cells at a rate of 0.2 mL/min at 48.3 °C for several hours until a stable baseline was obtained, and then 0.05% (w/v) MLG in 20 mM sodium acetate buffer (pH 5.5) was introduced into the flow cells at the same rate and temperature. Changes in frequency (Δf/n) and dissipation (ΔD) were recorded for different overtones (n). The large dissipation changes (ΔD > 5 ×

Chemical fractionation of grass cell walls suggests that MLG is tightly associated with cellulose and arabinoxylan (AX).12 Several functional roles have been suggested for MLG in primary walls, including coating cellulose microfibrils,9a forming gel-like matrixes in the wall,13 tethering cellulose microfibrils to form a load-bearing network,2b,9b,14 and providing a source of metabolizable energy.10b Although the structure of MLG is well-characterized, much less is known about how it interacts with other components of the cell wall matrix. This is especially true of the interactions between cellulose and MLG, which are essential for understanding the state of the cell wall matrix. This study characterizes the binding of MLG to cellulose in vitro and probes its role in the mechanical properties of isolated coleoptile walls using enzymatic digestion and wall creep assays.

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EXPERIMENTAL METHODS

Materials. The following polysaccharides and enzymes were purchased from Megazyme (Ireland): mixed-linkage glucan from barley (cat. no. P-BGBM); xyloglucan (XyG) from tamarind seed (cat. no. P-XYGLN); water-soluble arabinoxylan (AX) from wheat with 38% arabinose and 62% xylose (cat. no. P-WAXYI); insoluble arabinoxylan (inAX) from wheat flour with 36% arabinose, 51% xylose, and small amounts of glucose, mannose, and galactose (cat. no. P-WAXYI); (1,4)-β-D-mannan from ivory nut (cat. no. P-MANIV); arabinan from sugar beet (cat. no. P-ARAB); pectic galactan from potato (cat. no. P-PGAPT); (1,3)-β-D-glucan (cat. no. P-PACHY); and endo-(1,3)(1,4)-β-glucanase from Bacillus subtilis (EC 3.2.173; cat. no. E-LICHN). Glucuronoarabinoxylan (GAX) with an arabinose/4O-methyl-glucuronic acid/xylose ratio of 1:2:11 was isolated from spruce.15 Avicel (PH-101) was obtained from FMC BioPolymer (cat. no. 9004-34-6). To remove any residual soluble sugars associated with Avicel prior to addition of polysaccharides, 10 mg of Avicel was sequentially washed three times in 10 mL of 20 mM sodium acetate buffer (pH 5.5) for 30 min and centrifuged at 10 000g for 5 min to pellet the Avicel. For the binding of MLG to cellulose in vitro, we used two sources of cellulose: Avicel and regenerated cellulose (RC). Avicel is a form of microcrystalline cellulose prepared from wood by ball milling and extraction with hot acid. The resistant material remaining after extraction includes crystalline cellulose microfibrils. RC has the same chemical composition as microcrystalline cellulose but is amorphous and nonmicrofibrillar.16 Ultrapure water (Milli-Q Gradient A-10, Milli-Q, 18.2 MΩ·cm, < 5 ppb organic impurities) was used for all experiments. Trimethylsilyl cellulose (TMSC, degree of substitution = 2.71) was synthesized as previously described.17 Binding Isotherms. The time course for the binding of MLG to cellulose was analyzed by the depletion method. Two-hundred microliters of 0.5% (w/v) MLG solution was added to 1 mg of Avicel, and the mixture was diluted to a total volume of 1 mL with sodium acetate buffer (20 mM, pH 5.5). The samples were incubated and shaken on a temperature-controlled shaker (Multi-Therm, Benchmark Scientific) at 1500 rpm at 40 °C for 0.25, 0.5, 1, 2, 4, 8, 16, 20, and 32 h. After incubation, the samples were centrifuged for 10 min at 13 000g to pellet the Avicel. The amount of sugar in the supernatant was quantified by phenol/sulfuric acid assay,18 with glucose as the standard. The binding of MLG to cellulose, inAX, (1,3)-β-D-glucan, and (1,4)-β-D-mannan was also analyzed by depletion isotherms. In brief, variable amounts of MLG were added to buffer (20 mM sodium acetate buffer, pH 5.5) containing a fixed amount of binding substrate. For a standard MLG binding isotherm, 10, 25, 50, 100, 150, 200, or 250 μL of a 0.5% (w/v) MLG solution was added to 1 mg of Avicel, and the mixture was diluted to a total volume of 1 mL with sodium acetate buffer (20 mM, pH 5.5). The mixture was incubated and shaken on a temperature-controlled shaker (Multi-Therm, Benchmark Scientific) at 1500 rpm at different temperatures (22, 40, 60, and 80 °C). After incubation, samples were centrifuged for 10 min at 13 000g to pellet the binding substrate. The amount of sugar in the supernatant 1728

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10−6) necessitated Voigt-based viscoelastic modeling22 for the calculation of the mass, thickness, and viscoelastic parameters of the adsorbed films. The adsorption curves from multiple overtones (n = 7, 9, 11, and 13) were used for the viscoelastic modeling of the adsorbed layer to estimate the thickness (hf) and two other viscoelastic parameters (shear viscosity, ηf, and elastic shear modulus, μf) using the software package QTools 3.0.5 (Q-Sense). Values of thickness (hf) and density (ρf) of the adsorbed layer were not independent variables, and the density (ρf) was set to 1050 kg/m3 for the estimation of thickness (hf). For simplicity, we assume the density (ρf) to be constant throughout the adsorption process. Thus, for a user-specified density, fits for an effective thickness, elastic shear modulus, and viscosity can be found. For binding competition experiments, RC surfaces were modified by preadsorption of XyG and GAX from 20 mM sodium acetate buffer (pH 5.5). The sensor coated with a RC film was placed into the QCMD flow cells, and water was introduced at a rate of 0.2 mL/min at 48.3 °C for several hours until a stable baseline was obtained. XyG or GAX (0.05%, w/v) in sodium acetate buffer (20 mM, pH 5.5) was then introduced into the flow cell at the same rate and temperature as water, and Δf/n and ΔD were recorded simultaneously. The adsorption time required about 30 min until steady values in frequency and dissipation profiles were obtained. Lastly, water was reintroduced into the flow cells to remove reversibly bound molecules. The adsorption processes were monitored by QCM-D, and the resulting surfaces carrying the preadsorbed layers (XyG or GAX) were used for the experiments described below for MLG adsorption. These surfaces are referred to hereafter as XyG- or GAX-coated RC surfaces. Then, MLG (0.05%, w/v) in sodium acetate buffer (20 mM, pH 5.5) was introduced into the flow cell with XyG- or GAX-coated RC surfaces at the same rate and temperature. The adsorption of MLG from sodium acetate buffer (20 mM, pH 5.5) onto modified RC surfaces led to the formation of dissipative layers (ΔD > 5 × 10−6). For such MLG layers, the Sauerbrey equation is not valid for the calculation of the mass of the adsorbed layers, so the adsorption curves from different overtones (n = 7, 9, 11, and 13) were fit with the Voigtbased viscoelastic model. Atomic Force Microscopy (AFM) Measurements. The resulting MLG layers were dried in an oven at 60 °C overnight and then imaged with an MFP-3D-Bio atomic force microscope (MFP-3DBIO, Asylum Research) in tapping mode. The matrix polysaccharides could have potentially collapsed onto the surface of the RC as a result of the dehydration, but because the RC-coated surface was thoroughly washed with water prior to drying in the oven, there would be no changes in the comparative features. AFM images were collected under ambient conditions using a silicon tip (OMCL-AC 160TS, Olympus Corp.). The reported roughness estimates are rms values determined from the entire 2 × 2 μm2 scan areas. Wall Extension. Wheat coleoptiles (Triticum aestivum L. cv. Pennmore) and maize coleoptiles (Zea mays) were prepared as described by Li et al.23 Seeds were grown in the dark (3 days for maize and 4 days for wheat) at 27 °C. The coleoptiles were frozen at −80 °C and prepared for extension measurements. The samples were abraded with carborundum (to allow penetration of solutions), cut to ∼1 cm in length, boiled for 15 s in dH2O to deactivate native enzymes, and pressed between glass slides with a mass of 700 g for 10 min to remove cell fluids. Prepared coleoptile samples were clamped in a constant force extensometer (10 g for wheat and 20 g for maize) in 20 mM sodium phosphate buffer (pH 6.6). Wall extension was measured with a constant load extensometer as described by McQueen-Mason et al.24 In subsequent experiments, coleoptiles were digested with 20 U endo(1,3)(1,4)-β-glucanase in 200 μL of 20 mM sodium phosphate buffer (pH 6.6) after the extension baseline was stable. In addition to the buffer and enzyme treatment, some coleoptiles were also pretreated with sodium hydroxide (30 mM for wheat and 60 mM for maize) to facilitate wall loosening. After the baseline stabilized, the walls were washed extensively and treated with 20 U of endo-(1,3)(1,4)-βglucanase (200 μL of 20 mM sodium phosphate buffer pH 6.6). Determination of MLG in Maize and Wheat Coleoptiles. Fresh frozen 3 day old maize coleoptiles and 4 day old wheat

coleoptiles were ground under liquid nitrogen, pre-extracted with phenol/acetic acid/water (PAW),19 and lyophilized. To determine the amount of MLG in the coleoptiles, the samples were fractionated using the following treatments: (1) 50 mM CDTA (21 °C, pH 7.5) for 6 h, centrifuged (10 min at 900g), and washed with ddH2O (3×), and supernatants were collected and dialyzed; (2) 50 mM Na2CO3 containing 20 mM NaBH4 (1 °C) for 16 h, centrifuged (10 min at 900g), and washed with ddH2O (3×), and supernatants were collected; (3) 0.1 M KOH containing 20 mM NaBH4 for 2 h at 1 °C, centrifuged (10 min at 900g; repeated 2×), and washed with ddH2O (3×), and supernatants were collected; and (4) 6 M KOH containing 20 mM NaBH4 for 2 h at 1 °C, centrifuged (10 min at 900g; repeated 3×), and washed with ddH2O (6×), and supernatants were collected. The 0.1 M KOH and 6 M KOH soluble fractions were neutralized with acetic acid, dialyzed exhaustively against ddH2O at 4 °C, and lyophilized. The fractions were digested with 20 U of endo(1,3)(1,4)-β-glucanase in 1 mL of 20 mM sodium phosphate (pH 6.6) for 20 h. The reducing sugars in the digests were quantified by the phydroxybenzoic acid (PAHBAH) assay.25 Samples were suspended in 5% PAHBAH in 0.5 M HCl combined with 0.5 M NaOH and boiled for 5 min, and the absorbance was read at 410 nm. The 6 M KOH fraction was used to determine the total cell wall MLG. Additionally, 3 day old etiolated maize coleoptiles and 4 day old etiolated wheat coleoptiles (described above) were digested with 20 U of endo-(1,3)(1,4)-β-glucanase for 2 h at 26 °C in 1 mL of 20 mM sodium phosphate (pH 6.6). Sodium hydroxide-extracted samples were also digested with endo-(1,3)(1,4)-β-glucanase, and coleoptiles walls were pretreated with sodium hydroxide (30 mM for wheat and 60 mM for maize) for 20 min, washed extensively with buffer, and digested with the above enzyme. The digests were analyzed by the PAHBAH assay25 and HPAEC−PAD.

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RESULTS Binding of MLG to Cellulose. MLG bound to cellulose (Avicel) in vitro under the conditions previously used for binding of XyG to cellulose.26 An initial rapid phase of binding was followed by a slow phase that continued for at least 32 h (Figure 1). The basis for the slow phase of binding is not clear, but it may stem from slow diffusion of MLG into the finer crevices and pores of the Avicel particles, which are large aggregates nominally 50 μm in diameter. The effect of the slow binding can be seen in binding isotherms plotted at different time points after the rapid phase is completed (Figure 2). The apparent plateau in binding, as a function of initial MLG

Figure 1. Time course of MLG binding to Avicel in 20 mM sodium acetate buffer (pH 5.5) at 40 °C. The bars indicate standard error (n = 4). 1729

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increasing temperature and prolonged time were not due to a temperature-dependent change in Avicel structure: Avicel was heated and agitated for 2, 4, and 6 h at 60 °C, MLG was added, and the resultant solution was incubated for 2 h at 22 °C. The binding isotherm matched the binding isotherm of MLG incubated for 2 h at 22 °C (Supporting Information Figure 1). These results indicate that the increase in temperature altered the binding dynamics of MLG rather than the properties of the Avicel. To determine if the fine structure of the MLG bound to Avicel differed from unbound MLG, we examined the oligosaccharide profile of the bound and unbound MLG using endo-(1,3)(1,4)-β-glucanase digestion for 20 h at 60 °C. HPAEC−PAD analysis showed no significant difference in the oligosaccharide profiles (Supporting Information Figure 2). The ratio of trisaccharides to tetrasaccharides for the control MLG in solution and MLG bound to Avicel was similar (2.4:1 and 2.1:1, respectively; Supporting Information Figure 2). This shows that MLG that binds to cellulose is not enriched in oligosaccharides containing longer (1,4)-β-glucosyl repeating units (less than 5% according to Supporting Information Figure 2). Kinetics of MLG Adsorption on RC. The adsorption kinetics of MLG on RC were studied using QCM-D. The adsorption time for MLG solution was 1 h until the reintroduction of water, at which point the values of the frequency and dissipation did not return to baseline (Figure 4). This result confirms that MLG irreversibly binds to cellulose. The rate of adsorption was proportional to the concentration of MLG applied. The adsorption of MLG led to the formation of soft and dissipative layers on the cellulose films. For such layers, the Sauerbrey equation is not valid for the calculation of the mass of the adsorbed layers, and Voigt-based viscoelastic modeling is required. With the Voigt-based model, the adsorbed amount (ΓQCM‑D) was calculated as 18 mg/m2 for 0.05% MLG adsorption onto RC surfaces. The corresponding elastic shear modulus was 1.42 × 105 N/m2 and the shear viscosity was 1.11 × 10−3 N·s/m2 based on the modeling using the 7th to 13th overtone (Table 1). The amount of MLG in the solution did not significantly affect the elastic shear modulus, which ranged from 1.4 × 105 to 1.5 × 105 N/m2, and the shear viscosity, which ranged from 1.05 × 10−3 to 1.11 × 10−3 N·s/ m2. The thickness of the MLG coating on the RC increases with the increasing concentration of the solutions (15 nm in 0.01% MLG solution to 18 nm in a 0.1% MLG solution) (Table 1). The QCM-D results indicate that the MLG coatings on RC surfaces are relatively thick hydrogels and that the thicknesses are dependent on the solution concentration of the MLG. In addition, MLG irreversibly binds to both the amorphous RC surface and microcrystalline Avicel. Avicel/MLG Interaction with Other Cell Wall Polysaccharides. Binding isotherms of MLG onto other insoluble cell wall polysaccharides, including (1,3)-β-glucan, inAX, and (1,4)-β-mannan, were also investigated for 20 h at 60 °C. We found that MLG bound to (1,3)-β-glucan with a binding capacity of 18 μg/mg and to inAX with a binding capacity of 11 μg/mg (Supporting Information Figure 3). MLG did not bind to (1,4)-β-mannan. Thus, although the binding of MLG is not limited to cellulose, the polymer does not interact with all wall polymers. A number of polysaccharide binding competition experiments were performed. Several matrix polysaccharides, including arabinans and galactans (neutral pectins) as well as

Figure 2. Kinetics of MLG binding to Avicel from 20 mM sodium acetate buffer (pH 5.5) at 40 °C. The graph demonstrates the micrograms of bound MLG versus the amount of total sugar available in solution. The blue line represents micrograms of MLG bound at 20 h, the black line represents micrograms of MLG bound at 4 h, the red line represents micrograms of MLG bound at 2 h, and the green line represents micrograms of sugar released when the supernatant was removed, fresh buffer was added, and the bound MLG and Avicel were agitated for 20 h at 40 °C. The bars indicate standard error (n = 4).

concentration, after 2 h of incubation was 5 μg/mg of Avicel, increasing to 15 μg/mg of Avicel at 4 h and 28 μg/mg of Avicel at 20 h. When the supernatant was removed and fresh buffer was added, the bound MLG was not solubilized, indicating that MLG adsorption is irreversible. We also examined binding isotherms as a function of temperature: the binding maximum at 40 °C was 28 μg/mg of Avicel for 20 h. When the experiment was performed at 22 °C, the capacity for binding decreased to 6 μg/mg of Avicel for 20 h and increased to 44 μg/mg of Avicel at 80 °C for 20 h (Figure 3). The increases in MLG binding associated with

Figure 3. Temperature effect on MLG binding to Avicel with 20 mM sodium acetate buffer (pH 5.5) for 20 h. The graph demonstrates the micrograms of bound MLG versus the amount of total sugar available in solution. The blue line represents micrograms of MLG bound at 80 °C, black line represents micrograms of MLG bound at 60 °C, green line represents micrograms of MLG bound at 40 °C, and red line represents micrograms of MLG bound at 22 °C. Bars indicate standard error (n = 4). 1730

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Figure 4. Representative time-dependent Δf/n (A) and ΔD profiles (B) for different concentrations of MLG (w/v), 0.01 (blue), 0.025 (green), 0.05 (black), and 0.1% (blue), from 20 mM sodium acetate buffer (pH 5.5) at 48.3 °C absorbed on RC. Arrow indicates the addition of ddH2O. Curves correspond to the 5th overtone, and arrows indicate where solutions were switched.

Table 1. Summary of the Thickness, Adsorbed Amount, and Viscoelastic Parameters Obtained from Voigt-Based Model Using the 7th to 13th Overtonea

a

concentration of MLG

thickness hf

ΓQCM‑D

elastic shear modulus

shear viscosity

(%)

(nm)

(mg/m2)

μf × 10−5 (N/m2)

ηf × 103 (N·s/m2)

0.1 0.05 0.025 0.01

18.0 ± 1.2 16.7 ± 0.06 17 ± 0.6 15 ± 0.6

19 ± 1.2 17.5 ± 0.06 18 ± 0.6 16 ± 0.6

1.5 ± 0.2 1.42 ± 0.03 1.4 ± 0.1 1.4 ± 0.1

1.08 1.11 1.08 1.05

± ± ± ±

0.02 0.006 0.01 0.006

Values are given as the mean ± standard error (n = 3). A film density (ρf) of 1050 kg·m−3 was used for the modeling

Figure 5. Binding interaction (inhibition or promotion) between cell wall polysaccharides and MLG to Avicel with 20 mM sodium acetate buffer (pH 5.5) at 60 °C for 4 h. The graph shows the relative percent difference in the amount of bound sugar (arabinan, galactan, AX, and XyG) between the control standard curve binding to Avicel and the standard curve of the binding to Avicel with prebound polymer. (A) Binding inhibition of MLG to Avicel with prebound polysaccharides and (B) inhibition/promotion of the binding of cell wall polysaccharides to Avicell with prebound MLG. Bars indicate standard error (n = 4).

Subsequent binding of AX and XyG were unchanged, whereas the binding of arabinan and galactan was increased by 40% (Figure 5B). Monosaccharide analysis of the unbound supernatant from the competition experiment contained only the second polymer, not the prebound polymer, indicating that the second polysaccharide did not displace the prebound polymer (Supporting Information Figure 8). The inhibition of the binding of MLG provides insights into the construction of the primary cell wall because MLG binds to cellulose with XyG and AX in the wall. Inhibition of MLG Adsorption to RC with Other Cell Wall Polysaccharides. The kinetics of MLG adsorption onto

soluble AX and XyG (hemicelluloses), were examined for their effect on the binding of MLG to Avicel. Homogalacturonan (from citrus) was also tested, but it did not bind to Avicel (data not shown). The adsorption of MLG was inhibited by the presence of prebound cell wall polysaccharides (Figure 5A). XyG had the most significant inhibitory effect, reducing the binding capacity of MLG by 69% (Figure 6A; Supporting Information Figure 4B). AX reduced MLG binding by 36%, and the neutral pectins likewise blocked MLG binding by 17 to 18% (Figure 5A, Supporting Information Figures 5−7). It is noteworthy that prebinding of MLG to Avicel did not inhibit subsequent binding of other cell wall polysaccharides. 1731

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Figure 6. Representative time-dependent Δf/n (A) and ΔD (B) profiles for MLG from sodium acetate buffer (20 mM, pH 5.5) with concentration of 0.05% adsorption onto RC and modified RC surfaces, including XyG- and GAX-coated RC surfaces, at 48.3 °C. Arrow indicates the addition of ddH2O. Curves correspond to the 5th overtone, and arrows indicate where solutions were switched.

Table 2. Summary of the Thickness, Adsorbed Amount, and Viscoelastic Parameters Obtained from Voigt-Based Model Using the 7th to 13th Overtonea thickness hf

a

surface

(nm)

MLG bound to RC MLG bound to XyG-coated RC MLG bound to GAX-coated RC

16.7 ± 0.06 19 ± 0.6 13 ± 0.6

elastic shear modulus

shear viscosity

−5

ηf × 103 (N·s/m2)

μf × 10

2

(N/m )

1.42 ± 0.03 0.11 ± 0.03 1.31 ± 0.08

1.11 ± 0.006 0.73 ± 0.02 1.00 ± 0.006

Values given are the mean ± standard error (n = 3). A film density (ρf) of 1050 kg·m−3 was used for the modeling

experiments, we clamped coleoptiles in a constant force extensometer, added endo-(1,3)(1,4)-β-glucanase (20 mM sodium phosphate, pH 6.6), and measured the resulting extension response. The enzyme failed to induce wall extension (Figure 8A), yet parallel digestions with this enzyme released 12 to 13% of the MLG in the walls of maize and wheat coleoptiles (Figure 9). To make the wall more susceptible to digestion, the coleoptiles were pretreated with sodium hydroxide (30 mM for wheat and 60 mM for maize) to solubilize lightly bound matrix polymers, which promoted the enzymatic digestions to 18 to 19% of MLG (Figure 9); however, the endo-(1,3)(1,4)-β-glucanase still failed to induce wall extension (Figure 8B). HPAEC−PAD analysis confirmed that (1,3)(1,4)-β-glucan oligosaccharides were produced by enzyme digestion. Thus, although endo-(1,3)(1,4)-β-glucanase does digest MLG in the walls of these coleoptiles, it does not result in wall extension, suggesting that MLG does not act as a wall tether.

RC surfaces with prebound XyG and GAX was also monitored by QCM-D. MLG adsorption was reduced by more than 50% for the XyG-coated RC surface (Figure 6). The thickness of the MLG bound to the modified RC increased with the preadsorption of XyG and decreased with the preadsorption of GAX (Table 2). The fitting data showed a much thinner MLG layer on the GAX-coated RC surface. A stronger interaction between MLG and GAX leads to a more compact packing of MLG onto the GAX-coated surface than the larger molar mass XyG, which exhibits a thicker layer (∼20 nm; Table 2). The presence of GAX on RC surfaces does not significantly affect the viscoelastic properties of MLG layers compared to MLG adsorption onto bare RC surfaces, whereas adsorption of MLG onto a XyG-coated RC surface led to the formation of a softer and thicker MLG layer (Table 2). The surface layers were also examined by AFM: RC layers coated with GAX or XyG were rougher than those coated with MLG (Figure 7). AFM images showed that the adsorption of hemicelluloses onto RC roughened the surface, with a rms roughness value of 1.3 nm for the GAX-coated RC, 1.5 nm for the XyG-coated RC, and 1.0 nm for the MLG-coated RC. The roughened GAX-coated RC surface exhibits obvious aggregates, which become less distinct after MLG adsorption (Figure 7). However, there is no significant morphological change after MLG adsorption onto XyG-coated RC. The QCM-D results and the AFM images for the binding of MLG to GAX- and XyG-coated RC surfaces show that both hemicelluloses inhibit the subsequent adsorption of MLG onto RC and that MLG interacts differently with GAX and XyG. Function of MLG as a Cell Wall Tether. To test the hypothesis that MLG tethers cellulose microfibrils, we examined the ability of endo-(1,3)(1,4)-β-glucanase to promote cell wall extension, as has been previously demonstrated for expansins and some endo-1,4-β-glucanases.24,27 For these

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DISCUSSION The ability of MLG to bind to cellulose has been inferred from wall extractions,9a,12 but until now, it has not been tested experimentally. Like some other hemicelluloses, including XyG,26a,28 AX,29 and mannan,29c,30 we have shown that MLG binds irreversibly to cellulose in vitro. The in vitro conditions are a simplified artificial probe of the complex nature of the MLG interaction with cellulose and other matrix components. These results are consistent with the idea that MLG binds to the surface of cellulose microfibrils in the grass primary cell wall. The weight ratios of MLG adsorption in vitro to cellulose (1.5−4.4% w/w) for Avicel at 4 and 20 h is very similar to the reported values for XyG (1.3%) for Avicel31 and for pea XyG26a (1−5%) bound to other forms of cellulose. 1732

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together with the overall slow adsorption suggest that the process is enthalpy-driven. This temperature response is similar to the binding of xylan to cellulose.29a,32 The increased binding of GAX to cellulose at higher temperatures is mediated by the formation of aggregates,29a,33 which creates a thick layer on cellulose surfaces. Although GAX aggregates are formed from side-chain interaction,29a MLG is unbranched and thus aggregation must occur through backbone interactions. MLG aggregation and gel formation is likely facilitated by the high molecular weight and asymmetrical conformations of the polymer.6,34 Rheologically, MLG has been shown to act as random-coil polysaccharide in solution.7,34,35 Galactomannans also form random coils in solution, but gelling has not been demonstrated.36 The self-affinity shown by MLG6,34 suggests that this polysaccharide binds not only to cellulose surfaces but also to MLG already associated with cellulose, forming a thicker MLG layer. This is supported by QCM-D results, which indicated that MLG forms a thick hydrogel on amorphous RC. Because of the nature of the RC surface, it is still unknown whether MLG forms a gel over several microfibrils or attaches to a single microfibril. We also found the thickness of the MLG layer increases with the increasing concentration of MLG in solution. Using QCM-D, Paananen et al.33 also found that GAX formed an elastic layer on cellulose surfaces. However, the GAX adsorption curve did not reach a plateau for at least 3 h, whereas the adsorption of MLG reached its plateau in approximately 1 h with QCMD. The relatively fast adsorption of MLG onto RC measured is due to the surface adsorption of the polymer, whereas the bulk adsorption of MLG to Avicel is a much slower process. This is influenced by the available surface area of Avicel, which is a material composed of roughly spherical particles. These particles are dense aggregates with many internal surfaces. Binding adsorption for xylan and XyG is related to the surface area of the cellulose microfibrils.37 When MLG binding is examined on RC surfaces, a flat and smooth surface layer is formed, and the binding levels off rapidly. The relatively slow binding of MLG to Avicel compared to RC may

Figure 7. AFM height images of morphological changes before and after MLG adsorption onto (A) RC, (B) XyG-coated RC, and (C) GAX-coated RC. R represents the rms roughness determined from the entire 2 × 2 μm2 scan area. Values given are the mean ± standard error (n = 3).

Although our results do not provide direct evidence of the physical mechanism of the MLG−cellulose interaction, the increased binding of MLG to cellulose as temperature increases

Figure 8. Lack of endo-(1,3)(1,4)-β-glucanase-induced extension of heat-inactivated wheat and maize coleoptiles. The arrow indicates the addition of the endo-(1,3)(1,4)-β-glucanase (100 U/mL). The small spike in the extension rate is a purely mechanical effect from the addition of enzyme. (A) Coleoptile walls treated with endo-(1,3)(1,4)-β-glucanase (curves are means of 15 measurements) and (B) coleoptiles pretreated with sodium hydroxide for 30 min (30 mM for wheat coleoptiles and 60 mM for maize coleoptiles) followed by the addition of 20 U of endo-(1,3)(1,4)-βglucanase (curves are means of 16 measurements). 1733

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Figure 9. Sugar released by endo-(1,3)(1,4)-β-glucanase digestion of wheat and maize coleoptiles, determined by PAHBAH assay, and presented as micrograms of MLG released per milligram of coleoptiles. Bars indicate standard error (n = 3).

(w/w) binding of arabinan and 1.4% (w/w) binding of galactan. The binding of galactan and arabinan inhibited the binding of MLG by 17 and 18%, respectively, which is less than the inhibition of the hemicelluloses examined. This supports the hypothesis that pectin side chains, including arabinan and galactan, bind to cellulose microfibrils, although not as strongly as the hemicelluloses (e.g., XyG).31 The binding of galactan and arabinan increased by 40% with the presence of prebound MLG on Avicel; the long stretch of (1,4)-glucosyl units in the MLG polymer39 may provide areas for the neutral pectins to bind and/or the presence of a thick MLG layer may promote adsorption. These results indicate that MLG competes with other hemicelluloses for cellulose binding sites and could form a layered structure with MLG between cellulose and other cell wall polysaccharide bound to MLG. Although we have shown that MLG can bind to cellulose and other matrix polysaccharides, the functional role of this polymer in the wall remains unknown. To test the hypothesis that MLG acts as a physical tether between cellulose microfibrils, MLG was digested in coleoptiles using a specific endo-(1,3)(1,4)-βglucanase. Only 12 to 13% of the MLG was removed from the wall of the wheat and maize coleoptiles, which is significantly less than the amounts reported in maize coleoptile walls.9a However, significant amounts of the most accessible MLG were removed. If the MLG was tethered to cellulose microfibrils (rather than simply coating them), it would have been removed with the enzyme digestion. This supports previous results from maize coleoptile epidermis CW where MLG remains associated with the cellulose after extraction of most of the GAX by 1 M NaOH. Residual MLG after digestion displays a pattern of removal from the outside in, with the bulk of it remaining in the CW near the plasma membrane.9a Removal of XyG from cucumber hypocotyl cell walls with a XyG specific endoglucanase (approximately 45%) also did not cause wall extension.27c However, the hypocotyls walls did undergo extensions in responses to an enzyme that specifically hydrolyzes both XyG and cellulose. Large mechanical effects were seen when the enzyme digested less than 1% of the total wall XyG. Like XyG, the bulk of MLG in the cell wall does not act as a mechanical tether in the growing cell wall of the grasses. The role of MLG in the cell wall is still unknown, but the in vitro results and the fractionation results9a suggest that MLG could act as a coating layer to protect the microfibrils or a

reflect the differences in the relative accessibility of glucan surfaces in these samples. The formation of a hydrogel MLG layer on the cellulose surface supports the hypothesis that MLG acts as a gel-like matrix between cellulose microfibrils in the primary cell wall.13 The functions of gel-like polysaccharides in the cell wall include wall hydration, increasing porosity, promoting cell-to-cell adhesion, and regulating the access of wall-loosening proteins. In addition to the other functions of gel-like polysaccharides, MLG also interacted with other cell wall polysaccharides and binds to both AX and (1,3)-β-glucan when they are in the insoluble form. In binding competition experiments, pretreatment of Avicel with either XyG or GAX blocks subsequent binding of MLG. This is in agreement with XyG binding properties, where xylan was found to reduce the relative binding activity of pea XyG by 53%.26a Similarly, we determined that AX reduced the in vitro binding of MLG to cellulose by 36%. The QCM-D adsorption profiles show that there are smaller changes in the frequency and dissipation for MLG adsorption onto XyG-coated RC than for GAX-coated RC surfaces, indicating a stronger inhibition by XyG. On the basis of the Voigt-based viscoelastic modeling, a thinner MLG layer was formed on GAX-coated RC, which is in agreement with the observation that the stronger interaction between MLG and GAX leads to a more compact packing of MLG onto GAX-coated RC. Evidence of association between GAX and MLG has been reported in maize coleoptile cell walls9a and barley endosperm.38 Although pretreating cellulose with certain hemicelluloses can reduce subsequent MLG binding, pretreating cellulose with MLG neither blocks the subsequent binding of XyG or AX nor promotes hemicellulose adsorption onto cellulose. This is similar to the findings of Hayashi et al.26a in which lichenan (a form of MLG from Cetraria islandica) did not affect the binding of pea XyG to cellulose. These observations may result from XyG and AX binding to stretches of (1,4)-β-linked glucosyl residues4 in the MLG chain. Both of the neutral pectins, arabinan and galactan, inhibited the subsequent binding of MLG to Avicel. The reverse, however, is not true: the binding of both polymers increased when Avicel was pretreated with MLG. Previous reports that neutral pectic polysaccharides are able to bind to cellulose surfaces in vitro31 are confirmed by our results showing 2.3% 1734

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detection; inAX, insoluble arabinoxylan; MLG, mixed linkage glucan; NaOAc, sodium acetate; PAHBAH, p-hydroxybenzoic acid; QCM-D, quartz crystal microbalance with dissipation monitoring; R, roughness; RC, regenerated cellulose; RMS, root-mean square; TMSC, trimethylsilyl cellulose; U, unit of activity; XyG, xyloglucan

coating layer that acts as an adhesive for cellulose−cellulose interactions or cellulose−matrix polymer interactions. It is also possible that its primary biological role is not mechanical but metabolic (i.e., as a readily mobilized sugar source).10b,40

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CONCLUSIONS The observed binding of MLG to cellulose and other polysaccharides extends our understanding of the cell wall architecture of the grass cell wall. MLG, XyG, arabinan, galactan, and the different forms of xylans all compete for the same binding sites on cellulose. MLG forms a thick hydrogel when adsorbed onto a cellulose surface, where it could function as a transient attachment surface for other matrix polysaccharides during periods of rapid expansion. The results of cell wall creep assays indicate that enzyme-accessible MLG does not tether cellulose fibrils into a load-bearing network, but we cannot exclude a possible mechanical role for the nonaccessible MLG. It is also possible that two or more MLG domains exist in the cell wall: one that binds directly to the cellulose microfibril and one that controls intermolecular interactions.

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(1) (a) Pauly, M.; Keegstra, K. Plant cell wall polymers as precursors for biofuels. Curr. Opin. Plant Biol. 2010, 13, 304−311. (b) Cosgrove, D. J. Growth of the plant cell wall. Nat. Rev. Mol. Cell Biol. 2005, 6, 850−861. (c) Bergander, A.; Salmén, L. Cell wall properties and their effects on the mechanical properties of fibers. J. Mater. Sci. 2002, 37, 151−156. (d) Carpita, N. C.; Gibeaut, D. M. Structural models of primary cell walls in flowering plants: Consistency of molecular structure with the physical properties of the walls during growth. Plant J. 1993, 3, 1−30. (2) (a) Fincher, G. B. Revolutionary times in our understanding of cell wall biosynthesis and remodeling in the grasses. Plant Physiol. 2009, 149, 27−37. (b) Buckeridge, M. S.; Rayon, C.; Urbanowicz, B.; Tine, M. A. S.; Carpita, N. C. Mixed linkage (1→3),(1→4)-β-Dglucans of grasses. Cereal Chem. 2004, 81, 115−127. (c) Vogel, J. Unique aspects of the grass cell wall. Curr. Opin. Plant Biol. 2008, 11, 301−307. (3) (a) Carpita, N. C. Structure and biogenesis of the cell walls of grasses. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1996, 47, 445−476. (b) Trethewey, J. A. K.; Campbell, L. M.; Harris, P. J. (1→3),(1→4)β-D-Glucans in the cell walls of the Poales (sensu lato): An immunogold labeling study using a monoclonal antibody. Am. J. Bot. 2005, 92, 1660−1674. (c) Urbanowicz, B.; Rayon, C.; Carpita, N. Topology of the maize mixed linkage (1→3),(1→4)-β-D-glucan synthase at the golgi membrane. Plant Physiol. 2004, 134, 758−768. (4) Woodward, J. R.; Fincher, G. B.; Stone, B. A. Water-soluble (1→ 3),(1→4)-β-D-glucans from barley (Hordeum vulgare) endosperm. II. Fine structure. Carbohydr. Polym. 1983, 3, 207−225. (5) (a) Staudte, R. G.; Woodward, J. R.; Fincher, G. B.; Stone, B. A. Water-soluble (1→3),(1→4)-β-D-glucans from barley (Hordeum vulgare) endosperm. III. Distribution of cellotriosyl and cellotetraosyl residues. Carbohydr. Polym. 1983, 3, 299−312. (b) Woodward, J. R.; Phillips, D. R.; Fincher, G. B. Water-soluble (1→3),(1→4)-β-Dglucans from barley (Hordeum vulgare) endosperm. I. Physicochemical properties. Carbohydr. Polym. 1983, 3, 143−156. (6) Bohm, N.; Kulicke, W. M. Rheological studies of barley (1→3) (1→4)-β-glucan in concentrated solution: Mechanistic and kinetic investigation of the gel formation. Carbohydr. Res. 1999, 315, 302− 311. (7) Lazaridou, A.; Biliaderis, C. G.; Micha-Screttas, M.; Steele, B. R. A comparative study on structure−function relations of mixed-linkage (1→3),(1→4) linear β-D-glucans. Food Hydrocolloids 2004, 18, 837− 855. (8) Wilson, S. M.; Burton, R. A.; Doblin, M. S.; Stone, B. A.; Newbigin, E. J.; Fincher, G. B.; Bacic, A. Temporal and spatial appearance of wall polysaccharides during cellularization of barley (Hordeum vulgare) endosperm. Planta 2006, 224, 655−667. (9) (a) Carpita, N. C.; Defernez, M.; Findlay, K.; Wells, B.; Shoue, D. A.; Catchpole, G.; Wilson, R. H.; McCann, M. C. Cell wall architecture of the elongating maize coleoptile. Plant Physiol. 2001, 127, 551−565. (b) Labavitch, J. M.; Ray, P. M. Structure of hemicellulosic polysaccharides of Avena sativa coleoptile cell walls. Phytochemistry 1978, 17, 933−937. (10) (a) Gibeaut, D. M.; Carpita, N. C. Tracing cell wall biogenesis in intact cells and plants selective turnover and alteration of soluble and cell wall polysaccharides in grasses. Plant Physiol. 1991, 97, 551−561. (b) Roulin, S.; Buchala, A. J.; Fincher, G. B. Induction of (1→3,1→4)β-D-glucan hydrolases in leaves of dark-incubated barley seedlings. Planta 2002, 215, 51−9. (11) (a) Kim, J. B.; Olek, A. T.; Carpita, N. C. Cell wall and membrane-associated exo-β-D-glucanases from developing maize

ASSOCIATED CONTENT

S Supporting Information *

MLG oligosaccharides profiles from the digestion of bound and unbound MLG to Avicel; MLG adsorption curves to (1,3) βglucan, insoluble arabinoxylan, and (1,4) β-mannan; binding competition absorption curves between XyG and MLG polymers onto Avicel, arabinan and MLG polymers onto Avicel, galactan and MLG polymers onto Avicel, and AX and MLG polymers onto Avicel; and monosaccharides profiles of the supernatants are shown for the inhibition of MLG binding to Avicel by matrix polysaccharides. This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]; Phone: 814-865-3752. Author Contributions

All authors have approved the final version of the manuscript. S.N.K and D.J.C designed the research; S.N.K and X.Z. performed the research; S.N.K, X.Z., A.R.E, and D.J.C analyzed the data; P.G. and G.T. provided the glucuronoarabinoxylan; and S.N.K., X.Z., A.R.E., and D.J.C. wrote the paper. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by The Center for Lignocellulose Structure and Formation, an Energy Frontier Research Center funded by the U.S. Department of Energy, Office of Science, and Office of Basic Energy Sciences under award no. DESC0001090. The work at the Wallenberg Wood Science Center was funded by the Knut and Alice Wallenberg Foundation, which is greatly acknowledged for their financial support of G.T. and P.G. We acknowledge Liza Wilson with Dionex, Edward Wagner for assistance with the creep assays, and Eric Roberts for his assistance editing.

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ABBREVIATIONS AFM, atomic force microscopy; AX, arabinoxylan; GAX, glucuronoarabinoxylan; HCEC−PAD, high-performance anion-exchange chromatography with pulsed amperometric 1735

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